THE AGGLUTINATION REACTION 297 



enough to be of service in diagnosis (as in plague, for example), 

 or there are certain technical difficulties which make it inapplicable 

 (tuberculosis, cholera), while in still others a diagnosis can be con- 

 veniently reached in an even more direct manner (as by the isolation 

 and cultivation of the offending microorganism), etc. 



To give a general idea of the technical method of procedure it will 

 be best to describe the reaction as it is applied to the diagnosis 

 of typhoid fever, i. e., the Widal reaction proper. 



The Widal Reaction. TECHNIQUE. Microscopic Method. A small 

 amount of blood (5 to 10 drops) is collected in a little glass tube or 

 in one of the capsules pictured in Plate III. The serum is separated 

 by centrifugation and a drop diluted in the white mixing pipette 

 accompanying the hemocytometric counting chamber in the pro- 

 portion of 1 to 20. From this, subdilutions of 1 to 40, 1 to 80, 1 to 

 160 are prepared with the aid of the same pipette, normal salt 

 solution being used as diluent in all cases. Four slides are then 

 ringed with vaseline, and into each little chamber a drop of the 

 diluted serum is placed together with a drop of a typhoid culture in 

 bouillon, not more than twenty-four hours old. The resultant 

 dilutions will then be 1 to 40, 1 to 80, 1 to 160, 1 to 320. A cover- 

 glass is adjusted so as to be in contact all around with the vaseline, 

 as also with the drop in the central chamber. The specimens are 

 immediately examined with the middle power of the usual micro- 

 scopic outfit (1/6 B. & L.; No. 6 or 7 Leitz), and discarded if any 

 large clumps of bacteria are seen. Should this be the case, it is well 

 to make new mounts with a culture that has been centrifugalized 

 for a minute or two, the supernatant fluid only being used, in which 

 no clumps will be found. If the mount is satisfactory, it is set aside 

 and reexamined at the expiration of half an hour. If the reaction 

 is positive, all the bacilli will be found motionless at the expiration 

 of this time, and gathered in clumps of variable size (Fig. 15). This 

 will be the case at least in the lowest dilutions, while in the higher 

 ones it may be necessary to w r ait until another half -hour has expired. 

 The higher the dilution in which complete clumping may be obtained 

 the greater is the diagnostic significance of the reaction. Ordi- 

 narily, complete clumping at the end of half an hour in a dilution 

 of 1 to 40 is sufficient; if, however, the question of paratyphoid 

 enters into consideration, the result in the higher dilutions should 

 be considered. As the typhoid and paratyphoid bacilli carry certain 



